Doxorubicin (DOX) is one of the most effective chemotherapeutic agents used for cancer treatment, but it also causes systemic inflammation and serious multi-organ side effects and muscle dysfunction in many patients. The loss of muscle strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. However, it is unknown whether inflammation induced cell death, known as pyroptosis (a distinct form of cell death, compared to apoptosis, and necrosis) occurs in DOX induced muscle toxicity and dysfunction (DIMT). Our preliminary data shows that treatment with DOX induces pyroptosis in Soleus muscle (SM), Sol 8 cells which is inhibited by exosomes derived from embryonic stem cells (ES-exos). We also observed increase in pro-inflammatory M1 macrophages, toll-like receptor 4 (TLR4), formation of NLRP3 inflammasome and pyroptotic specific caspase 1, IL-1?, and IL-18 post-DIMT in SM following DOX treatment. Mice treated with ES-Exos post-DMIT decreased M1 macrophages, TLR4, NLRP3 inflammasome and SM pyroptosis. Moreover, phenoswitching of M1 macrophages occurred which significantly increased anti- inflammatory M2 macrophages and reduced pyroptosis as well as adverse SM remodeling. miRNA array data shows increase in miR200c in ES-Exos. Furthermore, our preliminary data shows significantly decrease in phosphorylated p38 and JNK in DIMT following ES-Exos treatment, suggesting involvement of p38/JNK pathway. Therefore, based on this compelling data, we hypothesize that ES-Exos ameliorate DOX-induced pyroptosis and associated adverse muscle remodeling by increasing anti-inflammatory M2 macrophages thereby improving muscle function post-DIMT. We propose to test this hypothesis through the following 3 specific aims.
Aim 1 : To determine if increased level of M1 macrophages induce TLR4 and activates inflammasome (NLRP3) mediated pyroptosis during short and long-term post-DIMT.
Aim 2 To demonstrate whether treatment with ES-Exos cause phenoswitching of M1 into M2 macrophages results in amelioration of pyroptosis and adverse SM remodeling. We will also determine the mechanisms of miR200c mediated p38 and JNK cell signaling in pyroptosis following ES-Exos treatment in DIMT. Additionally, we will examine the presence of broader range of miRNA specific for cell signaling, anti-pyroptotic, and anti-fibrotic pathways following ES-Exos treatment.
Aim 3 : To determine whether ES-Exos exert effects on anti-tumor efficacy of DOX in cancer while improving SM function. The proposed studies are hypothesis-driven, mechanistic, and should help define the impact of ES-Exos on M2 macrophage phenoswitching, pro- and anti-inflammatory cytokines, pyroptosis and apoptosis in the progression of DIMT. Also, we expect that ES-Exos would have no interference with the DOX-induced killing of cancer cells and inhibition of tumor growth. We believe that results obtained from these studies can easily be translated to the clinic as the cell free preparation of exosomes will not form teratomas, which is common occurrence with the use of human and mouse ES cells.
Doxorubicin is a wonderful anti-cancer drug. Unfortunately, a major side effect of this drug is causing a muscle dysfunction in patients suffering from cancer in the U.S. and around the world. Doxorubicin induces inflammation by attracting M1 macrophages is now becoming a considerable cause of development and progression of muscular dysfunction and fatigue. We report as a preliminary data that presence of M1 macrophages causing immune mediated cell death called pyroptosis and adverse muscle remodeling. We have generated ES-Exosome from ES cells which could be an excellent therapeutic option as these cell free components don't form teratoma and are capable to attenuate pyroptosis and adverse muscle remodeling is proposed in the grant. Overall if successful, ES-Exos will provide protection in DOX induced muscle toxicity by inhibiting pyroptosis and adverse muscle remodeling at short and long term of the disease. Overall if successful ES-Exos could have broad therapeutic option treating in doxorubicin induced toxicity in related diseases as well as in various other muscle dysfunctions.
|Merino, Hilda; Singla, Dinender K (2018) Secreted Frizzled-Related Protein-2 Inhibits Doxorubicin-Induced Apoptosis Mediated through the Akt-mTOR Pathway in Soleus Muscle. Oxid Med Cell Longev 2018:6043064|